Mechanism and regulation of riboflavin uptake by human renal proximal tubule epithelial cell line HK-2.
Riboflavin (RF), a water-soluble vitamin, is essential for normal cellular functions, growth, and development. Normal RF body homeostasis depends on intestinal absorption and recovery of the filtered vitamin in renal tubules. The mechanism and cellular regulation of the RF renal reabsorption process, especially in the human situation, are poorly understood. The aim of this study was therefore to address these issues, using a recently established human normal renal epithelial cell line, HK-2, as a model. Uptake of RF by HK-2 cells was found to be 1) linear with time for 5 min of incubation and occurring with minimal metabolic alterations, 2) temperature dependent, 3) Na+ independent, 4) saturable as a function of concentration [apparent Michaelis constant (K(m)) of 0.67 +/- 0.21 microM and maximal velocity (Vmax) of 10.05 +/- 0.87 pmol.mg protein-1.3 min-1], 5) inhibited by structural analogs and anion transport inhibitors, and 6) energy dependent. Protein kinase C-, protein kinase A-, and protein tyrosine kinase-mediated pathways were found to have no role in regulating RF uptake. On the other hand, a Ca2+/calmodulin-mediated pathway appeared to play a role in the regulation of RF uptake by HK-2 cells via an effect on the Vmax, as well as on the apparent K(m) of the RF uptake process. The uptake process of RF was also found to be adaptively regulated by the level of the substrate in the growth medium, with the effect being mediated through changes in the apparent K(m) and the Vmax of the uptake process. These results demonstrate that RF uptake by the human-derived renal epithelial cell line HK-2 is via a carrier-mediated system that is temperature and energy dependent and appears to be under the regulation of a Ca2+/calmodulin-mediated pathways and substrate level in the growth medium.
Kumar
C K
CK
Department of Medicine, University of California School of Medicine, Irvine 92697, USA.
Yanagawa
N
N
Ortiz
A
A
Said
H M
HM
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D001692:Biological Transport; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D009570:Nitriles / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012256:Riboflavin / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D013449:Sulfonamides; D013696:Temperature; D020032:Tyrphostins
D004791:Enzyme Inhibitors; D007546:Isoquinolines; D009570:Nitriles; D013449:Sulfonamides; D020032:Tyrphostins; C079836:tyrphostin 25; D005576:Colforsin; D003994:Bucladesine; D002772:Cholera Toxin; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D015056:1-Methyl-3-isobutylxanthine; D012256:Riboflavin